Abstract: Several important problems remain that prevent use of pig organs to save human lives, especially for pig lungs and livers. When exposed to human blood (or transplanted into baboons), our current multi-gene pig lungs and livers rapidly trigger disappearance of platelets and white blood cells from the circulation, stimulate ?inappropriate? blood clotting within the blood vessels of the xenograft, and activate a vigorous inflammatory response. The overall goal of this Project is to refine our current ?Platform Regimen? (pig genetic changes, drug treatments) by evaluating additional rationally targeted, mechanism-based gene modifications and pharmacologic approaches. The Specific Aims of the Project will be undertaken in ex vivo models, where pig lungs and livers are ?perfused? with human blood, and in life-supporting in vivo baboon transplant models. In this Project, we will evaluate whether two novel gene modifications to the pig will address the remaining problems. One modification is expected to inhibit coagulation pathway amplification by ?tissue factor? (Aim 1a), and the other targets platelet adhesion via the platelet's ?GP1b? receptor (Aim 2). We predict that these genetic changes to the Platform pig will be partly successful, but may not completely solve the problems of platelet and neutrophil adhesion and inflammation. Planning for this outcome, we have strong evidence that specific ?adhesion molecules? are responsible for some or all of the platelet and neutrophil adhesion phenomena that we see with our current Platform pig genetics. We will therefore also test several carefully chosen, clinically applicable drugs, and measure their effects on the residual pig organ injury response (Aims 1b, and 1c). These clinically applicable pharmacologic interventions will both help us understand the remaining problems better (define the contribution of specific pathways to liver and lung xenograft injury), and allow us to study mechanisms of organ injury that may currently be masked by the pathways under study in this Project and in Project 1. The revised Platform regimen will be evaluated in Aim 3, where the life-supporting capacity of pig lung and liver grafts will be rigorously assessed, using an immunosuppressive strategy identified from Project 1. If justified by interval findings, alternative approaches described in Project 2 or anti-inflammatory strategies (primarily under study in Aim 1) are available, one or more of which may prove necessary to reproducibly protect pig livers and lungs from injury in a human blood environment or in baboons. Project 2 is thus likely to solve several of the most important known barriers to clinical use of pig lungs and livers. Together with complementary contributions from Project 1, knowledge gained from this Program is also likely to contribute significantly toward therapeutic clinical application of lung and other organ xenografts: to facilitate progress into clinical trials by providing an evidence base for rationally selecting the specific strategies to be included in treatment of the first recipients of pig organ xenografts.